Aquaculture 493 (2018) 37–50

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Aquaculture
journal homepage: www.elsevier.com/locate/aquaculture

Review

Larval rearing of mud crab (Scylla): What lies ahead T

a a,⁎ c d e
Khor Waiho , Hanafiah Fazhan , Emilia T. Quinitio , Juliana C. Baylon , Yushinta Fujaya ,
⁎ ⁎
Ghazali Azmieb, Qingyang Wua, Xi Shia, Mhd Ikhwanuddinb, , Hongyu Maa,
a
Guangdong Provincial Key Laboratory of Marine Biotechnology, Shantou University, Shantou 515063, Guangdong, China
b
Institute of Tropical Aquaculture, Universiti Malaysia Terengganu, 21030 Kuala Terengganu, Terengganu, Malaysia
c
Aquaculture Department, Southeast Asian Fisheries Development Center, 5021 Tigbauan, Iloilo, Philippines
d
Division of Biological Sciences, College of Arts and Sciences, University of the Philippines Visayas, 5023 Miagao, Iloilo, Philippines
e
Faculty of Marine Sciences and Fishery, Hasanuddin University, Makassar 90245, Indonesia

A R T I C LE I N FO A B S T R A C T

Keywords: The increasing global demand for mud crabs (genus Scylla) and threats to the wild populations highlight the
Aquaculture urgency of fully rearing them in captivity. Despite considerable progress in mud crab production, most crab
Larval rearing farms still rely heavily on wild-caught crablets and juveniles while the low and inconsistent success rates of
Mud crab larviculture remain as the main bottleneck impeding the development of mud crab aquaculture. Over the years,
Rearing parameters
numerous studies have been conducted to determine the optimum larval rearing parameters, the ontogenic
Scylla spp.
changes in digestive function and feeding behaviour, and the diets for different larval stages. These data,
however, are dispersed and not summarised to inform culture practices. This review provides an update on the
current progresses and to pinpoint the gaps in knowledge regarding mud crab larval rearing. We include all four
mud crab species under the genus Scylla, i.e. Scylla serrata, Scylla olivacea, Scylla tranquebarica and Scylla
paramamosain. Knowledge compiled in this review serves as an important guideline for prospective mud crab
larviculture. Future research should gear towards filling in the gaps in our knowledge to advance mud crab larval
rearing, thus fully incorporating mud crab into the aquaculture sector.

1. Introduction 1998; Ikhwanuddin et al. 2011; Alberts-Hubatsch et al. 2016; Fazhan

et al. 2017a).
Mud crab genus Scylla De Haan, 1833 (Brachyura: Portunidae) is an They inhabit intertidal mangrove forests with fluctuating salinity
economically important crustacean species that is widely distributed and support the livelihood of local fishery communities (Keenan et al.
throughout the Indo-West-Pacific region (Keenan et al. 1998). Its tax- 1998; Ikhwanuddin et al. 2011; Alberts-Hubatsch et al. 2016). Global
onomy has been controversial. Mud crabs were previously recognised capture production of Scylla was above 20,000 t in the last decade (FAO
as a single species, i.e. Scylla serrata (Forskål, 1775). Estampador (1949) 2018) but the global aquaculture production of mud crabs however,
was the first to report three different species (S. serrata, S. oceanica was below 15,000 t since 1980 until 2003, after which it increased
(Herbst, 1796) and S. tranquebarica (Fabricius, 1798)) and one variation exponentially to above 100,000 t with the inclusion of data from China
(S. serrata var. paramamosain) based on their variation in size, colour (Fishery Bureau of Ministry of Agriculture of China 2012) and has been
and shape. However, due to the unclear differentiation between species increasing steadily ever since (FAO 2018). It is expected that the growth
and variation, different authors had different taxonomic classifications in aquaculture sector will rise due to increasing demand from the global
of the genus Scylla (Serene 1952; Stephenson & Campbell 1959; Perrine market (Goldburg & Naylor 2005). However, sustainable aquaculture
1978; Quinn & Kojis 1987) until Keenan et al. (1998) revised this genus has yet to be achieved for Scylla species since crab farms still rely
into four distinctive species based on their morphological, morpho- heavily on wild-caught crabs for seed stock, fattening, and soft-shelled
metric and molecular differences, namely S. serrata, S. tranquebarica, S. crab production (Ewel 2008; Alberts-Hubatsch et al. 2016; Waiho et al.
olivacea (Herbst, 1796) and S. paramamosain Estampador, 1949. The 2016, 2017; Fazhan et al. 2017b). Large scale aquaculture of mud crabs
geographical distribution of S. serrata is the widest, covering the tropics is currently limited due to very low success rate in the hatchery pro-
and subtropical coastal area of Indo-West-Pacific region while the other duction of juveniles (Quinitio & Parado-Estepa 2008; Holme et al.
three species are more concentrated around the equator (Keenan et al. 2009a; Waiho et al. 2015). For the past years, attempts for mass rearing

⁎
Corresponding authors.
E-mail addresses: fazhanhanafiah@gmail.com (H. Fazhan), ikhwanuddin@umt.edu.my (M. Ikhwanuddin), mahy@stu.edu.cn (H. Ma).

https://doi.org/10.1016/j.aquaculture.2018.04.047
Received 22 January 2018; Received in revised form 20 April 2018; Accepted 25 April 2018
Available online 27 April 2018
0044-8486/ © 2018 Elsevier B.V. All rights reserved.
K. Waiho et al. Aquaculture 493 (2018) 37–50

Table 1
General developmental descriptions and the average body length (ABL) of Scylla larvae to crab instar (Ong 1964; Jantrarotai et al. 2006; Lumasag et al. 2007; Yi et al.
2009; Thirunavukkarasu et al. 2014).
Crab stage Description

Zoea (Z) 1 Sessile eyes; abdomen segment = 5; lateral spines present on abdomen segments = 2 & 3 (S. olivacea), 3–5 (S. serrata); ABL = 1.33 mm (S. serrata), 1.12–1.25 mm (S.
olivacea)
Z2 Stalked eyes; abdomen segment = 5; lateral spines more distinct; ABL = 1.63 mm (S. serrata), 1.47–1.65 mm (S. olivacea)
Z3 Abdomen segment = 6 (plus telson); observable pereopod buds; ABL = 2.09 mm (S. serrata), 1.90–2.17 mm (S. olivacea)
Z4 Elongated lateral spines; distinct pleopod buds at the ventral posterior end on segments = 2–6; ABL = 3.05 mm (S. serrata), 2.35–2.80 mm (S. olivacea)
Z5 Pleopod buds increase in size, with setae and are biramous; ABL = 4.05 mm (S. serrata), 3.35–3.80 mm (S. olivacea)
M First pereopod developed into cheliped; increase in carapace width and size; ABL = 1.79 mm (S. serrata)
CI Crab-like appearance; carapace margin serrated; ABL = 3.06 mm (S. serrata)

Note: Z1 = zoea stage 1; Z2 = zoea stage 2; Z3 = zoea stage 3; Z4 = zoea stage 4; Z5 = zoea stage 5; M = megalopa; CI = crab instar.

of juvenile seeds in the hatchery had been carried out in countries such In the eye formation stage, a pair of red eyes as short thin strip can be
as Vietnam (Nghia et al. 2007a), Japan (Hamasaki et al. 2011), and the observed. Then, the embryo undergoes differentiation of the thorax and
Philippines (Quinitio et al. 1999, 2001; Quinitio & Parado-Estepa abdominal regions. Heartbeat is detectable at a rate of < 200 beats per
2008). min once it reaches stage 9. The differentiation of the telson at the
Crustacean larvae are highly vulnerable to diseases during the in- abdomen and the appearance of chromatophores on the thorax also
itial developmental stages and require specific biotic and abiotic con- occur during this stage. During the final prehatch stage, the heartbeat
ditions for normal growth and development (Azra & Ikhwanuddin rate is > 200 beats per min and the overall zoeal body are more de-
2015). Commercial hatchery production of crustacean larvae relies on fined. The ratio of embryo:yolk is about 4:1 of the total egg volume. The
the optimisation of three interrelated factors: rearing conditions, dis- colour of the egg mass changes from light yellow to dark orange, then
ease and nutrition (Sorgeloos & Léger 1992; Nghia et al. 2007a). The becomes light greyish orange and dark grey before turning to black
sub-optimal condition of any one of these factors significantly affects prior to hatching. With a mean rearing temperature of 28–29 °C, the
the health and growth of larvae, and if left unattended, results in mass incubation period of S. serrata (10.3 ± 0.3 days) was significantly
mortality of larvae. Works on various aspects of mud crab larviculture longer compared to that of S. tranquebarica (8.7 ± 0.6 days) and S.
and larval rearing technology were reported throughout the years. olivacea (8.6 ± 0.2 days) (Ates et al. 2012). When examined in vitro,
However, the production of mud crab larvae remained unsatisfactory the duration of embryonic stages of S. paramamosain increases with the
and most hatcheries encounter inconsistent survival rates of Scylla zoea decrease in water temperature, with eggs incubated at 30 °C hatched
to crablet stages primarily due to bacterial and fungal infections (Bian & within 9 days whereas those incubated at 20 °C took almost a month to
Egusa 1980; Nakamura et al. 1995; Roza & Hatai 1999; Lavilla-Pitogo & hatch (Zeng 2007). Similar results were also reported when eggs of S.
de la Peña 2004; Quinitio & Parado-Estepa 2008; Dan & Hamasaki serrata were maintained in vivo at a range of incubation temperature
2015), and molt death syndrome during the transition from zoea 5 (Z5) (20 to 30 °C) (Hamasaki 2003). It is therefore recommended that the
to megalopa stage (Williams et al. 1999; Hamasaki et al. 2002; Holme incubation temperature of ovigerous females is maintained at 29–30 °C
et al. 2007; Quinitio et al. 2015; Pates et al. 2017). Another main ob- to promote embryonic development and hasten larvae hatching.
stacle in Scylla larviculture is the lack of suitable larvae diets (Quinitio The detailed description of larval stages has been reported for the
et al. 2001; Baylon et al. 2004; Ruscoe et al. 2004; Holme et al. 2006; two Scylla species, S. serrata (Ong 1964) and S. olivacea (Jantrarotai
Baylon 2009; Holme et al. 2009b; Quinitio et al. 2015). Most hatcheries et al. 2006). Identification of each larval stage was based on the dif-
currently rely heavily on live foods (e.g. Artemia and rotifers) that have ferences of their appendages, i.e. mandible, maxillule, maxilla in-
inconsistent nutritional values (Tucker 1992; Quinitio et al. 1999, cluding antennule, antenna, maxilliped and telson (Ong 1964;
2001; Quinitio & Parado-Estepa 2008; Baylon 2010) and could also Jantrarotai et al. 2006). In general, a mud crab larva goes through five
potentially introduce harmful pathogens into the culture (Bentzon-Tilia distinct stages: Zoea 1 (Z1) to Zoea 5 (Z5) (Table 1), each lasts ap-
et al. 2016). This review presents and analyses the current knowledge, proximately 3–5 days, and megalopa stage which lasts for 7–10 days
research trends and efforts of hatchery practices used in mud crab prior to molting to first crab stage (C1) (Ong 1964) (Table 2). Each zoea
larviculture, with a general emphasis on the larvae of all Scylla species, stage can be distinguished from each other based on the differences in
if available. The advantages and differences of each aspect were dis- the number of setation of their maxilla (comparison of S. serrata and S.
cussed, and recommendations for the optimal hatchery production of olivacea larvae in Jantrarotai et al. 2006). Body length during zoeal
mud crab larvae were suggested. The gaps and bottlenecks that need to stage increases by approximately 30% between each molt (Yi et al.
be overcome in the future were also identified to drive the Scylla lar- 2009). The larval stages of mud crabs are pelagic and dispersive. It was
viculture forward. postulated by Webley and Connolly (2007) that they start to return to
the nearshore coastal shelf zone once they reach megalopa stage. The
pelagic megalopae then metamorphose into benthic crablets and with
2. Embryonic and larval development the assistance of shore currents and changing tides, these crablets re-
turn to intertidal zones and grow into adulthood.
The ten-stage embryonic development of three Scylla species, i.e. S. In addition to the five zoeal stages, a sixth zoeal stage (Z6) was
serrata, S. tranquebarica and S. olivacea has been described in detail by observed in S. paramamosain when feeding conditions are unfavourable,
Ates et al. (2012). In general, precleavage and cleavage occur right after e.g. unsuitable live food (fed solely on rotifers at later larval stages),
egg extrusion, and the embryo enters multicell stage within 1 day. It insufficient feeding and prolonged starvation (Zeng et al. 2004). The
will then enter the intermediate multicell-gastrula stage where the main differences between larvae of Z5 and Z6 were the increase in body
formation of one or two yolk-free spaces occur. In the following gastrula length and number of setae at all body parts (Zeng et al. 2004). A si-
stage, the yolk-free space increases in size and forms a U-shaped band milar additional zoeal stage was also reported in the larvae of other
(germinal disc). Appendage buds are apparent as translucent globular brachyurans such as the red frog crab Ranina ranina (Linnaeus, 1758)
structures in yolk-free space during this stage. A significant decrease in (Minagawa & Murano 1993), grapsid crab Chasmagnathus granulata
yolk volume (15–20% reduction from 95% in intermediate multicell- Dana, 1851 (Pestana & Ostrensky 1995) and mud crab Rhithropanopeus
gastrula stage) and opacity is expected in the subsequent naupliar stage.

38
K. Waiho et al. Aquaculture 493 (2018) 37–50

Table 2
The duration to molting (days) of each zoea stage of Scylla.
Larval developmental Duration to molting
stage
S. tranquebarica (Days) S. tranquebarica (Days ± s.d.) S. olivacea (Days) S. olivacea (Days) S. serrata S. serrata (Days) S. serrata
(Days) (Days)

Z1 to Z2 4 3.6 ± 0.9 – – 4–5 3–5 2

Z2 to Z3 3 3.4 ± 0.5 – – 3–4 3–4 2
Z3 to Z4 3 3.2 ± 0.4 – – 3 3–4 3
Z4 to Z5 3 2.4 ± 0.5 – – 3 3–4 3
Z5 to M 3 7.0 ± 0.7 – – 4–6 3–5 3
M to CI 6 3.4 ± 0.5 – – 8–10 – 7
TOTAL 22 27–28 18–19 (Z1-Z5) 20–29 (Z1-Z5) 25–31 18–20 20
Reference Thirunavukkarasu et al. Zohri 2011 Jantrarotai et al. Jantrarotai et al. Motoh et al. Heasman & Yi et al.
2014 2006 2004 1977 Fielder 1983. 2009

Note: Z1 = zoea stage 1; Z2 = zoea stage 2; Z3 = zoea stage 3; Z4 = zoea stage 4; Z5 = zoea stage 5; M = megalopa; CI = crab instar; s.d. = standard deviation;
“-” = no data.

harrisii (Gould, 1841) (McConaugha 1982) when reared in suboptimal 3.1. Temperature and salinity
dietary conditions. It was postulated that the variability in develop-
mental pathways is a coping mechanism of the larvae by prioritising Temperature and salinity are considered to be the important factors
survival over growth when faced with external stress (Pestana & in mud crab larviculture and changes in either of these factors would
Ostrensky 1995; Giménez & Anger 2003; Zeng et al. 2004). In the influence the internal physiological processes of larvae, ultimately af-
nursery, the occurrence of the sixth zoeal stage strongly suggests that fecting their growth performance and survival (Hamasaki 2003;
the rearing conditions in the hatchery are not optimal. Nurdiani & Zeng 2007; Baylon 2010; Azra & Ikhwanuddin 2016).
Hill (1974) tested 61 combinations of salinity (0–35 ppt) and tem-
perature (5–36.5 °C) on the first zoeal stage of S. serrata (50–200 zoeae
3. Collection of zoea and rearing conditions
per treatment), with an exposure time of 24 h. He found that Z1 of S.
serrata were not tolerant to low salinity levels (almost 100% mortality
On average, a female can spawn up to 5 million eggs per batch and
when salinity of < 14 ppt at all temperatures except 10 °C). Active
about 80% of the eggs will hatch (Quinitio et al. 2001; Davis et al.
zoeae with survival rates of > 90% were recorded when subjected to
2004). Females in captivity were found to be less fecund compared to
salinity of > 17.5 ppt at 10–25 °C (except for those combinations that
wild ovigerous females, with their mean zoeae rate of
fell out of the > 90% survival curve) (Hill 1974). Hamasaki (2003),
7767 ± 4169 zoeae/g. weight and 9307 ± 1200 zoeae/g. weight, re-
however, noticed that Z1 were unable to molt to Z2 at 17 °C whereas
spectively (Maheswarudu et al. 2007). The number of offspring pro-
normal development until Z5 was found at 20–35 °C (Table 3).
duced by broodstocks varied greatly even if they were reared in the
Although tolerant to the fluctuations in temperature and salinity,
same pond (Quinitio et al. 2001), indicating that possible internal
mud crab larvae showed better survival and faster growth at an op-
factors such as age, genetics and health might play an important role in
timum range of 25–30 °C and 20–35 ppt (Table 3) (Jantrarotai et al.
the reproductive performance of females. Significant decrease in es-
2002; Hamasaki 2003; Nurdiani & Zeng 2007; Baylon 2010, 2011,
sential fatty acids (e.g. histidine, lysine, isoleucine, leucine and phe-
2013). In addition, faster development was observed at higher tem-
nylalanine) was reported in successively spawned Scylla larvae (second
perature for the larvae of both S. serrata (Hamasaki 2003; Nurdiani &
and/or third spawning) (Islam & Yahya 2016). In addition, successive
Zeng 2007; Baylon 2011) and S. olivacea (Baylon 2010). A shorter de-
third spawning also showed lower fertilization rates, decreased fe-
velopmental time is preferred as this may reduce the exposure time of
cundity, lower production of healthy phototactic larvae, and higher
larvae to other potentially adverse factors such as predation, physical
proportion of aborted eggs (Islam & Yahya 2016). This highlights the
stress, cannibalism of megalopae on zoea and food availability (Morgan
better quality of larvae hatched from first-spawned eggs, which should
1995; Quinitio et al. 2001; Baylon 2010). Delayed development was
be a factor to consider when using larvae for culture. To assess the
observed at salinities beyond the optimum range (i.e. 15, 40 and 45 ppt)
quality of the newly-hatched Z1, formalin stress test (3 h exposure to
and low temperatures (23 °C and below) (Table 3). Baylon (2011)
40 mg/L of formalin) is recommended (Quinitio et al. 2018). A low
reared S. serrata and S. olivacea larvae at different salinity and tem-
mortality rate of 0%–18% indicates that the batch of larvae is healthy
perature levels, and the larvae of these two species seem to have similar
and larval rearing is highly recommended. However, a mortality rate
responses. Parado-Estepa and Quinitio (2011) conducted a study to
of > 38% indicates that the larvae are of poor quality and further
compare salinity requirements of three Scylla species and found out that
rearing may be discontinued.
survival rates of S. olivacea and S. serrata zoeae to megalopa were not
Newly-hatched zoeae are recommended to be transferred to suitable
affected by salinity (8, 12, 16, 20, 24 and 32 ppt). That of S. tranque-
clean rearing tanks immediately after hatching to reduce chances of
barica, however, were significantly lower in higher salinity (24 and
bacterial infection in the spawning tank (Quinitio & Parado-Estepa
32 ppt). Interestingly, all three species showed lesser growth (in terms
2008). Owing to the phototactic nature of the newly spawned zoeae,
of body weight and internal carapace width) when reared in 32 ppt for
they may be collected by siphoning or direct scooping under a light
75 days continuously. Parado-Estepa and Quinitio (2011) also indicated
source at the water surface. The estimation of zoeae may be carried out
that S. serrata crab instars could tolerate a wider salinity ranges com-
during this stage prior to allocating them to different rearing tanks.
pared to the other two species.
Acclimatization should be carried out by placing the zoeae in a basin
The tolerance of mud crab zoeae to low temperature and wider
and float the basins in the larval rearing tanks for approximately
salinity range increased with growth (Baylon 2010, 2011). Approxi-
20–30 min if there is a temperature difference between the spawning
mately 95% of C1 juveniles survived at low temperature of 20 °C and
tank and the larvae rearing tanks (Nghia et al. 2007a; Shelley &
their salinity tolerance increased to 15–45 ppt and 5–45 ppt at mega-
Lovatelli 2011). Various external factors such as temperature, water
lopa stage and C1 stage, respectively (Baylon 2010). The enhanced
salinity, larval stocking density, the use of antibiotic or formalin, and
tolerance is probably due to the internal growth of the mud crab. The
tank colour should be optimized to allow for maximum larval survival.

39
K. Waiho et al. Aquaculture 493 (2018) 37–50

Table 3
Cumulative duration (days) and survival rate (%) of larval development from hatching to first crab stage (C1) of Scylla spp. reared under different temperature and/or
salinity combinations.
Species Temp. (°C) Salinity (ppt) Cumulative days (survival rate, %) to each stage Reference

Z2 Z3 Z4 Z5 M C1

S. serrata 17 34 - (0a) - (0a) - (0a) - (0a) - (0a) - (0a) Hamasaki 2003†

20 34 7.95 (91.7b) 14.15 (91.7b) 21.6 (85.0b) 28.85 (71.7b) 38.45 (50.0b) - (0a)
23 34 4.45 (95.0b) 8.45 (95.0b) 12.85 (95.0b) 18.35 (93.4b) 25.4 (41.7b) 41.8 (23.4b)
26 34 3.55 (98.4b) 6.4 (95.0b) 9.45 (91.7b) 12.85 (88.4b) 17.75 (81.7c) 35.35 (40.0b)
29 34 3.0 (98.4b) 5.0 (96.7b) 7.05 (95.0b) 10.05 (88.4b) 13.0 (83.3c) 56.2 (68.4c)
32 34 2.4 (95.0b) 4.4 (91.7b) 6.7 (85.0b) 8.8 (76.7b) 11.65 (61.7bc) 22.0 (36.7b)
35 34 2.7 (96.7b) 4.8 (88.3b) 7.35 (75.0b) 10.35 (66.7b) 18.0 (1.8a) - (0a)
S. serrata 25 15 5.7 12.6 17.3 – - (0f) – Nurdiani & Zeng 2007‡
25 20 4.6 7.8 12.3 16.8 22.6a (37.3abcd) 31.9
25 25 4.5 7.9 11.5 15.6 20.7abc (49.3ab) 33.0
25 30 4.9 7.8 11.8 15.9 20.6abcd (52.0ab) 36.0
25 35 5.1 8.5 12.5 16.8 21.4ab (54.7a) 35.7
28 15 3.3 6.7 10.6 14.2 23.0 (2.7ef) –
28 20 3.4 5.8 8.9 12.0 15.8e (50.7ab) 23.0
28 25 3.2 5.5 8.3 11.2 15.3e (42.7abc) 22.7
28 30 3.2 5.9 8.9 11.7 15.5e (30.1abcd) 24.0
28 35 3.3 6.5 9.3 12.1 16.2cde (28.2abcde) 25.3
31 15 3.8 6.9 9.4 – - (0f) –
31 20 3.3 6.3 9.3 12.5 16.9bcd (33.3abcd) 22.8
31 25 3.8 6.6 9.6 11.8 16.0e (37.3abcd) 23.2
31 30 4.0 7.2 9.7 12.5 16.7cde (28.0bcde) 24.2
31 35 5.0 7.5 10.0 13.1 16.9bcde (20.0cde) 26.2
34 15 3.0 – – – - (0f) –
34 20 3.1 4.9 – – - (0f) –
34 25 2.8 4.8 7.8 10.9 13.5e (13.3def) 20.0
34 30 3.1 5.4 8.5 11.6 16.1de (13.7def) –
34 35 3.2 5.8 9.1 12.1 18.5e (2.8ef) 24.5
S. serrata 20 0 – – – – – – Baylon 2010§
20 5 – – – – – –
20 15 – – – – – 22 (10c)
20 25 8 (70bc) 15 (53bc) 22.5 (18c) 32 (13d) 39 (26c) 55.5 (60ab)
20 35 8 (73ab) 14.5 (71b) 22 (32c) 31.5 (12d) 44.5 (49b) 62.5 (53ab)
20 45 – – – – – 21.5 (13cd)
26 0 – – – – – –
26 5 – – – – – –
26 15 – – – – – 10 (20cd)
26 25 3.5 (79ab) 7 (94a) 10 (88ab) 13 (57bc) 17.5 (89a) 26.5 (70ab)
26 35 3.5 (87a) 7 (96a) 10 (90ab) 13 (73ab) 17.5 (85a) 26.5 (57ab)
26 45 – 5.5 (71b) 9.5 (87ab) 14 (52f) – 26.5 (17cd)
32 0 – – – – – –
32 5 – – – – – –
32 15 – – – – – 7 (40bc)
32 25 2.5 (63bc) 5 (92a) 7.5 (89ab) 10 (82a) 13 (83a) 19.5 (83a)
32 35 2.5 (71bc) 5 (95a) 7.5 (93a) 10 (83a) 13 (82a) 20 (52ab)
32 45 4 (52c) 8 (40c) 11.5 (77b) 14.5 (49c) 17.5 (17c) 9 (43bc)
S. olivacea 20 0 – – – – – – Baylon 2011§
20 5 – – – – – –
20 15 – – – 10.5 (53c)§ 24 (26e)§ 35 (5d)§
20 25 9.5 (36c) 16.5 (29b) 24.5 (35bc) 33 (80b) 44 (80bcd) 57 (58bc)
20 35 9.5 (35c) 16.5 (33b) 24.5 (35bc) 34 (71b) 45 (68cd) 57.5 (53bc)
20 45 – – – – – –
26 0 – – – – – –
26 5 – – – – – –
26 15 – – – 5 (69b)§ 10 (84abcd)§ 17.5
(82ab)§
26 25 4 (66ab) 8 (79a) 12 (50a) 16 (91a) 21 (97ab) 27 (71abc)
26 35 4 (54b) 8 (70a) 12 (46ab) 16 (71b) 21 (56de) 28 (69bc)
26 45 – – – – – 9 (5d)§
32 0 – – – – – –
32 5 – – – – – –
32 15 5 (27c) 8 (27b) 12 (29c) 16 (76b) 19.5 (96ab) 25.5 (71abc)
32 25 4 (69a) 6.5 (86a) 9.5 (54a) 12.5 (92a) 16 (99a) 5 (93a)
32 35 4 (66ab) 6.5 (75a) 9.5 (50a) 12.5 (80b) 16 (88abc) 21.5 (82ab)
32 45 5 (26c) 7.5 (39b) 11.5 (31c) 15.5 (22d) – 26.5 (47c)
S. tranquebarica 20 0 – – – – – – Baylon 2013§
20 5 – – – – – –
20 15 – – – – – –
20 25 9.5 (35)c 21 (10)c 32.5 (9)c 43 (13)e – 11.5 (20)
cd §
20 35 8.5 (55)d 20 (2)d 31.5 (14.7)c 42 (30)d – 11.5 (13)
d§
(continued on next page)

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K. Waiho et al. Aquaculture 493 (2018) 37–50

Table 3 (continued)

Species Temp. (°C) Salinity (ppt) Cumulative days (survival rate, %) to each stage Reference

Z2 Z3 Z4 Z5 M C1

20 45 13.5 (0)¶ – – – – –
26 0 – – – – – –
26 5 – – – – – –
26 15 – – – – – 12 (13)
d§
26 25 4 (90)a 9 (76)a 13.5 (81)a 18.5 (71)b 26.5 (48)a 37 (67)ab
26 35 4 (91)a 9 (79)a 13.5 (71)ab 18.5 (76)b 27 (22)b 37.5 (60)b
26 45 6 (20)c 13 (42)b 19 (21)c 26 (45)c – –
32 0 – – – – – –
32 5 – – – – – –
32 15 – – – – – 10 (27)c§
32 25 2.5 (93)a 6 (70)a 8.5 (62)b 11.5 (87)a 17.5 (51)a 27.5 (78)a
32 35 2.5 (91)a 6 (80)a 8.5 (84)a 11.5 (91)a 17.5 (48)a 27.5 (67)ab
32 45 4 (82)a 10 (50)b 14.5 (19)c 19.5 (40)cd – 10.5 (13)d§
S. olivacea – 12 – – – – – 9.8 (57.1 bB) Jantrarotai et al. 2002
– 16 – – – – – 9.7 (95.2 aA)
– 20 – – – – – 10.3 (95.2 aAB)
– 28 – (37.6bB) – (18.5 bC) – (15.8 bC) – (14.5 bC) 23.6 (13.2 bC) –
– 30 – (51.8 aB) – (31.8 aC) – (35.6 aCD) – (30.1 aD) 24.2 (22.9 aE) –
– 32 – (30.1 bB) – (14.2 bC) – (11.0 bC) – (9.8 bC) 23.8 (8.3 bC) 11.3 (95.2 aA)
– 34 – (31.7 bB) – (13.3 bC) – (9.6 bD) – (8.9 bD) 24.0 (7.1 bD) –
– 36 – – – – – 13.5 (52.4 bB)
– 40 – – – – – 20 (9.5 cB)

Note: Data of duration and survival rate are expressed as mean; different superscript letters within the same row (uppercase) or column (lowercase) indicate
significant difference (P < .05) within the same experiment; “–” = no data.
†
The same set of larvae were monitored in the entire experiment and survival was taken at every zoeal stage up to crablet 1.
‡
The same set of larvae were monitored in the entire experiment but survival was taken only at crablet 1.
§
From the same batch, new larvae in every stage were used where development & survival was taken up to crablet 1.
¶
Larvae survived for 13.5 but died without molting to next stage.

thickening and mineralization of the integument as the zoea grow eating, coupled with the low immune system of newly hatched larvae
(Freeman 1993) protects them from sudden temperature changes makes them highly vulnerable (Verschuere et al. 2000). The use of
(Baylon 2010) while the formation of functional gills when they reach antibiotics is still prevalent in most crustacean hatcheries (e.g. mud
Megalopa stage (Felder et al. 1986) enables them to withstand extreme crabs and shrimps) due to their proven efficacy against Vibrio and other
salinities. infectious microorganisms (Diggles et al. 2000), which improves larval
survival (Gardner & Northam 1997; Azam & Narayan 2013). Without
3.2. Stocking density prophylaxis, luminescent bacteria (Vibrio spp.) were present and de-
tectable in S. serrata larvae rearing tanks with an increasing trend from
Stocking density during the fragile larval rearing stages of Scylla has day two onwards and subsequent onset of larval mortalities was pos-
immediate influence on the survival, growth, behaviour, water quality tulated to be related to the high bacterial load (Quinitio et al. 2001).
and food intake (Chambel et al. 2015). Setting the stocking density too Due to the small size of the larvae, prophylaxis treatments during larval
low (10 and 25 Z1/L) or too high (above 200 Z1/L) was reported to stages of Scylla are often incorporated directly into the rearing water
cause adverse effects for Scylla larvae in terms of larval survival and (de Pedro et al. 2007; Pates et al. 2017). Survival rates of larvae treated
metamorphosis (Baylon & Failaman 1999; Nghia et al. 2007a). A low with antibiotics (oxytetracycline, penicillin G or polymyxin B sulphate)
stocking density might result in excess food, thus polluting the rearing were found to be significantly higher than that of controls (Table 6).
medium, promoting bacteria growth and eventually causing mortality. The application of antibiotics, however, should be used with caution
On the contrary, setting the stocking density too high increases food because bacteria becoming increasingly resistant towards the ad-
competition among zoeae, introduces stress and impedes their overall ministered antibiotics (Schmidt et al. 2000) could adversely impact the
development (Nghia et al. 2007a; Chambel et al. 2015). In addition, cultured organisms, the environment and humans (see review by
from an economic point of view, the highest stocking density achiev- Cabello 2006). In addition, residual antibiotics in aquaculture species
able, without compromising crab's health and normal growth is pre- due to excessive use are proven to be transmittable to consumers, thus
ferred as this would maximize the usage of space during mass pro- potentially affecting their normal microbe flora and render them sus-
duction. Based on previous researches (Tables 4 & 5), stocking density ceptible to infection (Angulo et al. 2004). Although the usage of anti-
of zoeae at 100–150 Z1/L is recommended for Scylla spp. biotics (i.e. oxytetracycline and furazolidone) resulted in higher sur-
vival and faster growth, morphological deformities were found in both
larval (bent dorsal, rostral and furcal spines) and subsequent juvenile
3.3. Prophylaxis stages (fused lateral spines, asymmetrical abdomen, fused frontal
spines, depressed abdomen tip and gap between sternites) (Pates et al.
The larval stages of Scylla are highly susceptible to bacterial and 2017). Similar occurrence of deformities was reported in the megalopae
fungal infections, and protozoan infestation. If left untreated, often of temperate crab Pseudocarcinus gigas (Lamarck, 1818) due to the
resulted in mass mortality (Armstrong et al. 1976; Quinitio et al. 2001; prolonged use of antibiotics (oxytetracycline of ≥25 ppm) since larval
Quinitio & Parado-Estepa 2008). This is the case for most aquatic cul- stage I (Gardner & Northam 1997).
tured species as they are constantly surrounded by an environment that Apart from antibiotics, other chemicals such as formalin or tri-
gives equal opportunity for the growth of pathogens (Moriarty 1998). fluralin are also commonly used as prophylactic agents in shrimp and
The higher chances of exposure via contact and while drinking or

41
K. Waiho et al. Aquaculture 493 (2018) 37–50

Table 4
Stocking density (Z1/L) and survival rate (%) of Scylla spp. Zoea (Z) 1.
Species Stocking density (Z1/L) Final survival rate (%) Reference

S. paramamosain 50 27 (up to Z5) Djunaidah et al. 2001

75 39 (up to Z5)
100 63 (up to Z5) Nghia et al. 2007a
5 4 ± 6 (up to 22 d)
100 5 ± 4 (up to 22 d)
150 5 ± 1 (up to 22 d)
200 5 ± 1 (up to 22 d)
S. tranquebarica 75 51.8–55 (up to Z2) Maheswarudu et al. 2007
50.0–54.3 (up to Z2)
100
S. serrata 30–50 2.7 ± 0.8 (up to three- to five-day old megalopa) Quinitio et al. 2001

Table 5 of the probiotic bacteria against pathogenic bacteria was evident up to

The recommended stocking density for zoea (Z) 1. Z5. Another commercially available probiotics (PondPlus®A, Novozy-
Species Stocking density (Z1/L) Reference
mes®A)1 containing multispecies Bacillus was tried on S. paramamosain
larvae with great success (at concentrations of 0.5 and 1.0 g/50 L) in
S. paramamosain 100–150 Nghia et al. 2007a terms of higher larval survival, faster larval growth and development,
S. paramamosain 100 Djunaidah et al. 2001 and the absence of pathogenic green Vibrio at the end of larval culture
Scylla spp. 30–200 Shelley & Lovatelli 2011
S. serrata 50 Baylon & Failaman 1999
(Talib et al. 2017). However, these study on probiotics were conducted
S. serrata 80–100 Quinitio & Parado-Estepa 2008 in small containers using an average of 12,808 (Dan & Hamasaki 2015)
and 5000 (Talib et al. 2017) individuals per tank and in controlled
conditions. The beneficial effects of probiotics might not be as apparent
Table 6 in commercial-scale seed production as various conditions are complex
The survival percentage (%) of Scylla zoeae (Z) treated with prophylactic and hard to control. For example, during seed production in a large
chemicals. scale, culture of larvae and live foods (e.g. rotifer, Artemia and phyto-
Species Test Chemicals used Survival (%) Reference plankton) are done in the same rearing vessel and added subsequently
duration with various regiments as the larvae develop (Wang et al. 2005; Baylon
2009). The live foods act as potential pathogen carriers and studies
S. serrata Up to Z4 Control 5 ± 6.2 Azam &
have shown that the structural composition of the microflora in rearing
Control 7 ± 2.2 Narayan 2013
OTC (10 ppm) 25 ± 2.4 medium is constantly changing (Verdonck et al. 1997). One possible
OTC (25 ppm) 53 ± 4.1 solution to this problem is to administer probiotic via oral admission,
S. paramamosain Up to 22d Control 9 ± 5.0a Nghia et al. i.e. enrichment of live food. A study on the effect of Bacillus probiotic
Formalin (20 μL/ 11 ± 8.0ab 2007a (Bacillus licheniformis and B. subtilis, ratio = 1:1) on the growth and
L)
OTC (10 ppm) 21 ± 5.0b
survival of Pacific white shrimp, Litopenaeus vannamei (Boone, 1931)
S. serrata Up to Z5 Control 0 Brick 1974 revealed that larvae fed with probiotic-enriched live food (Artemia
p.B. 60 nauplii and rotifers) were significantly larger, heavier and had lower
(10 ppm) + US mortality rate (Jamali et al. 2015). Garrido-Pereira et al. (2014) also
p.G. 50
demonstrated that by administering probiotic directly into the rearing
(40 ppm) + US
water and via live feed, the larvae of marine finfish, Rachycentron ca-
Note: OTC = oxytetracycline; p.B. = polymyxin B sulphate; p.G. = penicillin nadum (Linnaeus, 1766) exhibited higher tolerance towards salinity
G; US = unfiltered seawater; different superscript letters indicate significant stress.
difference (P < .05) within the same experiment.

crab cultures (Williams et al. 1986; Gardner & Northam 1997). The 3.4. Tank colour
latter however, was banned by the European Union due to its toxicity to
The background colour of the rearing tanks plays an important role
aquatic organisms and bioaccumulation (European Union 2012). When
tested on Scylla larvae up to 96 h in each stage (Z1 to Z5), formalin (5 or in maintaining the general wellbeing of larvae and facilitate predation
in vision-oriented larvae by influencing the amount and spectrum of
10 μg/L) promote better larval survival and higher number of mega-
lopae compared to control (de Pedro et al. 2007). However, prolonged light that penetrates the rearing water column (Maciel & Valenti 2014).
Although it might be beneficial for hatcheries to use tanks of lighter
use of formalin as prophylactic treatment however, have been reported
to cause deformities and significantly increased molting mortality in background to contrast the dark coloured Scylla zoeae for ease in larval
monitoring, Rabbani and Zeng (2005) reported that S. serrata larvae
other crab species (Gardner & Northam 1997). Thus, periodical ex-
posure to formalin bath (5 or 10 μg/L) is recommended if prophylaxis preferred rearing tanks with dark coloured background, especially
black, as evidenced by their significantly higher survival rate, shorter
treatment is needed (Kaji et al. 1991; Hamasaki & Hatai 1993).
Another promising approach in the prophylaxis in Scylla larval developmental duration and more synchronized molting. In contrast,
larvae reared in white tanks had the lowest survival rate and longer
rearing is the application of probiotics. These are live microorganisms
that are advantageous to the host animal (Fuller 1989; Irianto & Austin developmental time. Similar results were observed in other crustacean
species, including the blue swimming crab, Portunus pelagicus (Lin-
2002; Kesarcodi-Watson et al. 2008; Dan & Hamasaki 2015) and added
to inhibit the growth of pathogenic bacteria in aquaculture environ- naeus, 1758) (Azra et al. 2012) and Amazon River prawn, Macro-
brachium amazonicum (Heller, 1862) (Maciel & Valenti 2014), with a
ments (Verschuere et al. 2000; Dan & Hamasaki 2015). Dan and
Hamasaki (2015) screened and isolated a strain of probiotic bacteria higher survival and maximum productivity in tanks with black
that work against necrosis-causing bacteria, thus overcome larval ne-
crosis symptoms and mass mortality in S. serrata. The inhibiting effect 1
The mention of brands does not indicate endorsement of the products.

42
K. Waiho et al. Aquaculture 493 (2018) 37–50

background in both studies. immunity of cultured organisms (Yaakob et al. 2014). Therefore, the
Although crustaceans are non-obligated visual feeders – able to feed conventional clear-water rearing method is now being substituted by
in total darkness based on random encounter or chemosensors the incorporation of microalgae into rearing medium, especially in the
(Minagawa & Murano 1993; Gardner & Maguire 1998), they do rely on aquaculture sector (van der Meeren et al. 2007; Neori 2011; Tendencia
their visual cues when available (Gardner & Maguire 1998). Apart from et al. 2015). With the aforementioned advantages of microalgae, it is
increasing prey visibility, light is also positively associated with can- therefore recommended to incorporate them into Scylla larval rearing.
nibalism – minimal cannibalism was found in dark coloured tanks
(Rabbani & Zeng 2005). It is also important to note that due to the 4. Nutrition
phototactic behaviour of most larvae, including that of Scylla spp.,
larvae in white tanks tend to aggregate and remain at the tank's bottom 4.1. Nutritional requirement
whereas those in black tanks were uniformly distributed throughout the
water column due to the greater light reflection in lighter-coloured Nutritional requirements may be viewed as the needed nutrient for
tanks at the bottom of the tank (Rabbani & Zeng 2005; Maciel & Valenti optimal growth and/or survival, or the minimum nutrient required to
2014). The reflection and immediate light source from above might maintain an organism alive or to fulfil a physiological role (Hamre et al.
cause disorientation and eventually negatively affect their feeding 2013). The nutritional requirement of larvae must be high and differ
(Rabbani & Zeng 2005). Additionally, the bottom-aggregation beha- qualitatively and quantitatively from that of juveniles and adults as
viour in light-coloured tanks will also increase the exposure of larvae to they undergo rapid growth, and dramatic morphological and physio-
potential infectious pathogens as most dead natural feed, uneaten food logical changes during these early life stages (Hamre et al. 2013).
and dead larvae accumulate at the bottom. Limited research has been conducted to investigate the nutritional re-
quirements of Scylla larvae and with the availability of a previous re-
3.5. Rearing system view regarding this topic by Holme et al. (2009b), this section of the
current review focuses primarily on studies conducted on Scylla larvae
Owing to its simplicity, most mud crab hatcheries reared larvae in to facilitate easier knowledge-transfer and to bring forward the latest
either clear water or microalgae-enriched systems with manual water discoveries on this topic.
exchange (Quinitio & Parado-Estepa 2008; Shelley & Lovatelli 2011; Proteins are made up of amino acids and are an essential ingredient
Thirunavukkarasu et al. 2014). Nghia et al. (2007a) reported that clear of growth for all animals (Holme et al. 2009b). Although research on
recirculating systems with biofilters and a water exchange rate of 100% the protein requirements of Scylla larvae has not been conducted, stu-
tank volume per 3–4 h were more conducive for Scylla larval rearing – dies done on juveniles were promising. Those fed with diets containing
higher survival rates at 15 and 18 days after hatch and lower ammonia 50% lipid-free casein (Sheen & Wu 1999; Sheen 2000), 32–40% protein
and nitrite levels, compared to clear water system with manual water (Catacutan 2002) or 45% crude protein (Unnikrishnan & Paulraj 2010)
exchange (30–50% daily). The advantages of recirculating systems were showed improved growth and survival. The high protein requirement
also reported in the larviculture of other aquaculture animals reported in juveniles highlights the importance of protein for the
(Blancheton 2000; Garrido-Pereira et al. 2014; Holan et al. 2014; growth and development of Scylla species, and it may be postulated that
Joaquim et al. 2014; Ballester et al. 2017). Further, less labour and Scylla larvae, being carnivorous and showing high protease activity
seawater are needed owing to the automated and water-recycling fea- since Z1 stage (Hong et al. 1995), may have a protein requirement si-
tures of the recirculating systems. Based on the positive results obtained milar or higher than in juvenile stages. Research in this area is war-
by Nghia et al. (2007a) using rearing systems of 30 and 100 L, large- ranted to determine the protein requirement and suitable protein
scale application of recirculating system in Scylla larviculture is pro- source of dietary protein for different stages of mud crab larvae.
mising. However, Nghia et al. (2007a) recommended that the rearing of Essential amino acids are an integral part of the dietary requirement
early stages (Z1 and Z2) larvae are more suitable in microalgae-en- of crustaceans (Holme et al. 2009b). The essential amino acids and non-
riched non-recirculating system. Recirculating system could be applied essential amino acids of day-old Scylla zoea was described by
at a later stage once the larvae are stronger (Z3 and above). This is to Peñaflorida (2004) by comparing with the essential amino acid profiles
mitigate the stress imposed by the water current of the recirculating of rotifers, Artemia and Acartia. Enrichment of rotifers with histidine,
system onto the fragile early crab larvae and also to prevent them from Artemia with leucine and tryptophan, and Acartia with arginine, leucine
getting caught on the mesh net of the recirculating system, damaging and valine are recommended to enhance larvae production.
the long spines on their carapaces. The importance of essential and non-essential fatty acids such as
The importance of incorporating microalgae in the rearing system highly unsaturated fatty acids (HUFA), docosahexaenoic acid (DHA),
was also highlighted in the study of Nghia et al. (2007a), with larvae eicosapentaenoic acid (EPA) and arachidonic acid (ARA) in live feed are
reared in clear recirculating systems manifested significantly lower shown to influence the survival, development and metamorphosis of
survival rate compared to those reared in algae-enriched recirculating mud crab larvae (Suprayudi et al. 2004a; Nghia et al. 2007b). By en-
and manual water exchange systems (Chlorella for Z1-Z3 stages, Chae- riching rotifers and Artemia with varying ratios and percentages of
toceros for Z4 stage onwards). The lower amount of ammonia and nitrite DHA/EPA and HUFA respectively, Nghia et al. (2007b) found that no
in recirculating systems (both with and without microalgae) than in HUFA content or high (50%) HUFA content but low DHA/EPA ratio
manual water exchange systems highlights the positive effect of bio- (0.6) led to delayed development and failure to metamorphose. In
filter in maintaining water quality. Larvae reared in clear recirculating contrast, Scylla larvae performed best in HUFA content of 30% and
systems also showed significantly lower larval stage index compared to DHA/EPA ratio of 4. Dietary ARA improved only the first metamor-
those reared in recirculating systems with microalgae (Nghia et al. phosis rate (from Z1 to Z2) but not the survival or development at zoeal
2007a). The benefits of introducing microalgae into rearing systems are stages of Scylla (Nghia et al. 2007b).
numerous. Aside from serving as food for live feed (zooplankton such as Crustaceans need cholesterol as a precursor for various important
Artemia and rotifer), microalgae are also known to aid in the stabili- physiological compounds including the production of sex hormones and
zation and quality improvement of the introduced rearing medium, molting hormones (Sheen 2000). Sterols are considered as essential
including higher dissolved oxygen and pH stabilization (Tendencia nutrients for crustaceans as they lack the ability to convert acetate to
et al. 2015), improve gut filling at the initiation of exogenous feeding sterol and have to secure it via dietary route (Kanazawa & Teshima
(van der Meeren et al. 2007), provide probiotic effects (Bergh et al. 1971; Sheen 2000). When fed with Artemia enriched with cholesterol at
1994; Haché et al. 2017), regulate bacterial growth (Krishnani et al. levels of 0 ul/l (free sterols = 0.61%), 5 ul/l (free sterols = 0.67%), and
2010; Cadiz et al. 2016) and enhance growth (Nghia et al. 2007a) and 10 ul/l (free sterols = 0.75%), S. serrata larvae showed better survival,

43
K. Waiho et al. Aquaculture 493 (2018) 37–50

intermolt duration and body size at C1 stage compared to those fed with of different zoea stages of Scylla larvae is needed for a better under-
20 ul/l (free sterols = 1.37%) of cholesterol (Suprayudi et al. 2012a). standing of their nutritional needs and optimization of their diets.
Thus, a minimum of 0.61% cholesterol is needed for the normal growth
and development of Scylla larvae and if Artemia is used as live feed, no 4.2. Ontogeny of feeding apparatus and feeding behaviour
additional enrichment is necessary as the cholesterol in the Artemia
nauplii alone is already sufficient (Suprayudi et al. 2012a). It is worthy Although knowing the nutritional requirement of Scylla larvae
to note that too much dietary cholesterol during larval (≥1.37%) would contribute to the optimization of diet and feeding regimes, it
(Suprayudi et al. 2012a) and juvenile stages (> 1.12%) (Sheen 2000) would be useless if the given diets were not ingested by the larvae due
may exert adverse effects on mud crab growth such as low survival, low to physical and/or behavioural constraints. Understanding of the
weight gain, prolonged intermolt period and smaller carapace width. feeding behaviour, food acquisition process and morphology of the
The exact mechanism resulting in adverse effects due to high level of feeding apparatus of Scylla larvae are essential for the formulation of
dietary cholesterol, however, is still unknown but is postulated to be a feed that would promote ingestion, digestion, and absorption.
nutrient-response characteristic which is toxicity-related (Mercer Feeding behaviour of Scylla Z1 suggests that telson is used to push
1982). water, along with any prey or food particles from underneath its ab-
Phospholipids play an important role in the structural composition domen towards its mouth (Maheswarudu et al. 2007). Non-food parti-
of cell walls and are involved in the absorption of sterol by acting as cles that are stuck onto the maxillules' setae are quickly removed with
surfactants for the emulsification of ingested lipid (Teshima 1997; the aid of two long denticulate setae. Abdomen and telson occasionally
Holme et al. 2009b). They are also the key component during molting exert gentle pressure on maxillules and maxillae, resulting in the tem-
by transporting cholesterol to different parts of the target tissue porary bending away (about 45 degrees) of furcal spines and setae and
(Conklin et al. 1983). Although crustaceans are being postulated to be assist in the passing on of food into the foregut (Maheswarudu et al.
able to synthesize phospholipids de novo (Sheih 1969), the synthesis 2007). Additional flaps by the abdomen direct food towards the mouth.
rate was slow and additional dietary phospholipid are needed for It takes approximately three minutes for food particles (50–55 μm) to be
maintaining normal growth and development, especially in the early digested as indicated by the gut movement of S. serrata Z1. This feeding
larval stages (Teshima et al. 1986). Among the proven positive effects of behaviour of Scylla larvae is consistent irrespective of the types of diet
dietary phospholipids on crustacean culture include enhanced growth (Maheswarudu et al. 2007).
and survival, improved molting rates and reduced molt death syndrome Generally, the feeding apparatus of brachyuran larvae involves the
(Conklin et al. 1980; Paibulkichakul et al. 1998; Gong et al. 2001). The mouthparts and the gastric mill. The mouthparts are further divided
most commonly used phospholipid in the aquaculture industry is in the into six paired appendages, in which three anterior pairs (mandibles,
form of soybean lecithin (Holme et al. 2009a). S. serrata larvae ex- maxillules and maxillae) are cephalic appendages and another three
hibited improved survival rate, intermolt period and larger carapace posterior pairs (first, second and third maxillipeds) are thoracic ap-
width (measured at the first crab stage) when fed with Artemia enriched pendages (Ng 1998; Lumasag et al. 2007). Located posteriorly at the
with DHA (25 ul) and soybean lecithin (0–40 ul), and Artemia enriched anterior chamber of the foregut, the gastric mill serves as a chewing and
with cuttlefish phospholipids (40 ul) (Suprayudi et al. 2012b). Higher grinding apparatus. The most notable difference in the morphology of
molting synchronicity index was also observed in treatments fed with mouthparts as larvae develop is the presence of teeth and denticles in
Artemia enriched with DHA and soybean lecithin at 20 ul and 40 ul the mandibles only during zoeal stages but not in megalopa. In addi-
respectively, and Artemia enriched with cuttlefish phospholipids tion, the simple and continuous foregut of early zoeae (Z1-Z3) develop
(Suprayudi et al. 2012b). Thus, a minimal amount of phospholipid, in and form two distinctive compartments when they reach Z4 or Z5
combination with essential fatty acids are required for the synchronized stages (Lumasag et al. 2007). As the width of mouth opening increases
growth and development of mud crab larvae. As proven by Suprayudi from 61 μm in Z1 to 95 μm, 129 μm, 179 μm and 209 μm in Z2, Z3, Z4
et al. (2012b), no additional DHA is required if cuttlefish phospholipids and Z5, respectively (Lumasag et al. 2007), appropriate screening of
were used as they are high in saturated fatty acids and essential fatty suitable feed particle size could be conducted, ensuring that only food
acids such as DHA, EPA and ARA (Reis et al. 2016). that could be ingested by larvae are given at a specific stage.
Although carbohydrates are thought to be non-essential in the diet The gut evacuation time of S. serrata larvae at different develop-
of crustaceans and that aquatic species are comparably less efficient in mental stages was determined by Serrano (2012b) to understand their
utilizing dietary carbohydrates (Shiau & Peng 1992). However, carbo- feeding and digestive strategies. In general, the gut evacuation time was
hydrates and lipids are still being incorporated into the feed of most shorter in early zoeal stages (approximately 80 min) than in megalopa
aquaculture animals including crustaceans since these are utilised first and first crab instar stage (120–135 min). A faster gut evacuation time
to produce energy, then proteins (Stuck et al. 1996; Johnston 2003). was also recorded when Z3 larvae to first crab instar were fed with live
From a feed developer's perspective, carbohydrate is preferred as it is feed compared to those fed with artificial feed (Serrano 2012b). The
the most economical dietary energy source (cost/kcal) (Shiau & Peng slower gut evacuation time in larvae fed with artificial diet is postulated
1992). Data on the suitable dietary carbohydrate levels of Scylla larvae, to be a result of insufficient enzyme (endopeptidase and exopeptidase)
however, is still unavailable and the limited studies on other crustacean activities (Serrano & Traifalgar 2012) and carnivorous crustacean
larvae (Penaeus Fabricius, 1798 and Homarus Weber, 1795) showed larvae are known to be unable to regulate their own enzyme levels in
wide variation in their required dietary carbohydrate levels (Kurmaly response to artificial diets (Jones et al. 1997). In addition, the change in
et al. 1989; Immanuel et al. 2003; Moe et al. 2004; Pedroza-Islas et al. feeding behaviour of Scylla larvae is obvious – from feeding on sus-
2004; Fiore & Tlusty 2005). It was observed that S. serrata juveniles pended particles in the water column during zoeal stages to looking for
were able to digest and fully utilize (91.6–95.8%) a wide range of food near the bottom during megalopa stage, which is closely related to
carbohydrates, especially from plant feedstuffs such as copra meal, their metamorphosis from planktonic larvae to clawed megalopae with
wheat flour, rice bran, corn meal and soybean meal (Catacutan et al. a more benthic nature (Genodepa 2003; Holme et al. 2006).
2003). Digestive enzyme profiles of S. serrata (Rutledge 1999) and the
spiny lobster Jasus edwardsii (Hutton, 1875) (Johnston 2003) suggest 4.3. Prey/particle size
that carbohydrates are essential in the diet of small juveniles as in-
dicated by the higher cellulase (in S. serrata), amylase and laminarinase It is important to consider the size of prey or food particles pre-
(in J. edwardsii) specific activity in smaller individuals. This shows that sented to any hatchery-reared organisms to maximize ingestion (Knauer
the requirement for dietary carbohydrate varies between development & Southgate 1999). Scylla larvae are postulated to employ raptorial
stages and future research on the carbohydrate nutritional requirement feeding method as in most caridean and homarid larvae (Jones et al.

44
K. Waiho et al. Aquaculture 493 (2018) 37–50

1997; Genodepa et al. 2004a). This indicates that they are able to Table 7
consume food particles with larger size ranges (Genodepa et al. 2004a). Common enrichment methods of rotifers and Artemia used in aquaculture.
The full digestion of feed, however, is still related to the mandibular Live feed candidate Enrichment method Fed larvae Reference
size of larvae. For example, with the mandibular size of about 61 μm
(Lumasag et al. 2007), approximately 80% of the S. serrata Z1 suc- Rotifers
Tetraselmis spp. S. serrata Davis et al.
cessfully digested veliger larvae (Pinctada fucata (Gould, 1850)) of size
- Brachionus Nannochloropsis spp. S. serrata 2005a, 2005b
50–55 μm (positive digestion indicated by the passing through the gut) plicatilis S. tranquebarica Suprayudi et al.
whereas none could consume pelagic copepods (Pseudodiaptomus spp. 2002
Herrick, 1884 and Labidocera spp. Lubbock, 1853) due to their large S. serrata Baylon 2009
size (500–1000 μm) (Maheswarudu et al. 2007). There was preference Dry Selco®

for bigger food particle size with increasing developmental stage of

- Colurella Ruscoe et al.
Scylla larvae. Genodepa et al. (2004a) demonstrated that S. serrata Z1 adriatica 2004
ingested more microbound diets of < 150 μm compared to those of Artemia franciscana
150–250 μm, whereas Z3 were able to ingest microbound diets up to nauplii
Super Selco® S. serrata Davis et al.
400 μm (highest ingestion in those of size 150–250 μm) although no
- EG® type Frippak™ CD2 Ultra S. serrata 2005b
statistical significant difference was observed in different feed particle - AF grade larval shrimp food Ruscoe et al.
sizes. The Z5 showed significant preference to microbound diets with 2004
feed particle size of 250–600 μm compared to those of 150–250 μm Artemia nauplii Oleic acid, EPA28G, S. serrata Suprayudi et al.
while megalopae significantly ingested more feed with particle size of DHA70G 2004b

400–800 μm (Genodepa et al. 2004a). This increase in preferred feed

Note: The mention of brands does not indicate endorsement of the products.
particle size is in positive correlation with the increase in width and
development of the mandibles (Lumasag et al. 2007), enabling larvae to
Enrichment of rotifers and Artemia are recommended and often
feed on larger prey more efficiently. Thus, the suitable particle size
carried out to ensure that they provide sufficient nutritional values to
range for Z1, Z3, Z5 and megalopae recommended are < 150 μm,
promote growth and maintain the general health of the larvae (Quinitio
150–250 μm, 250–600 μm and 400–600 μm respectively (Genodepa
et al. 1999; Davis et al. 2005a, 2005b). Various methods were used to
et al. 2004a). It is also worth mentioning that Scylla larvae were ob-
enrich either rotifers or Artemia and have been tested to be suitable for
served to only partially ingest their prey or food particle (Baylon et al.
mud crab larviculture (Table 7). Unenriched rotifers have minimal
2004).
nutritional value where the survival of the larvae was not significantly
better than that of starved larvae in a feeding experiment conducted on
4.4. Digestive enzymes
the giant freshwater prawn, Macrobrachium rosenbergii (de Man, 1879)
(Lovett & Felder 1988).
The larvae of Scylla spp. do not possess the anterior midgut di-
Based on the current findings, a combination of rotifers at 10–40
verticulae unlike some crustaceans (Serrano 2012a). Unable to rely on
individuals/ml (Quinitio et al. 2001; Baylon et al. 2004; Ruscoe et al.
the anterior midgut diverticulae to produce digestive enzymes as in
2004; Suprayudi et al. 2004b; Baylon 2009) and newly-hatched Artemia
penaeid larvae and their hepatopancreas are still not fully developed
nauplii at 0.5 individuals/ml (Ruscoe et al. 2004) are to be given once a
until megalopa stage (Serrano 2012b), Scylla larvae have to resort to
day at Z1-Z3 and only Artemia nauplii at 1.5 individuals/ml or in-
other methods such as increasing the gut passage time in order to digest
creasing density of 0.5 to 2 individuals/ml (Suprayudi et al. 2002))
and absorb more nutrients and energy from their diet (Jones et al. 1993;
when mud crab larvae reach Z3 or Z4 onwards (Suprayudi et al. 2002;
Serrano 2012b). A recent study by Serrano (2013) confirmed that
Davis et al. 2005a, 2005b; Quinitio & Parado-Estepa 2008; Baylon
amylase and leucine aminopeptidase (LAP) levels were low at mud crab
2009). The availability of rotifers during the early larval stages, espe-
Z1-Z3 while trypsin level was minimal at Z1-Z2 and megalopa stages. In
cially within the first hour of hatching to Z1 significantly improved the
the treatments with live feed (rotifers and Artemia), however, con-
survival of larvae (Mann et al. 1999). The inclusion of Artemia nauplii
siderable amount of exogenous LAP (64–80%) and trypsin activities
during the early larval stages are proven to improve metamorphosis
(37–43% at Z2 to C1) were recorded. These findings highlight the
rate and increase larvae size (chela length, carapace length and setae
contribution and importance of live feed as an integral source of exo-
bearing pleopods) (Suprayudi et al. 2002; Davis et al. 2005b). It is
genous enzymes for the development of mud crab larvae.
important to note that the added Artemia nauplii density should be kept
below 5 ind/mL to reduce unnecessary competition with the crab larvae
4.5. Diets used
for space and dissolved oxygen, and to minimize potential water fouling
from the contributed metabolites, which in turn could cause stress and
4.5.1. Live feed
even mortality to the crab larvae (Baylon et al. 2004). Due to their
The current larviculture of mud crab relies heavily on live feed due
lower nutritional values, rotifers may be removed from the mud crab
to beneficial factors including the ease of mass culture, slow swimming
larvae's diet after Z3 as no significant adverse effect on growth and
characteristics, free dispersal in water and availability in various sizes
survival was reported (Ruscoe et al. 2004; Baylon 2009). Baylon et al.
(Hill 1979; Verischele 1989).
(2004) reported that there is a reduction in rotifer intake and an in-
creasing consumption of Artemia as the mud crab larvae grow. After Z3,
4.5.1.1. Rotifers and Artemia. Their small size (approximately 0.5 μg
the presence of rotifers no longer affects the Artemia intake. Shift of
and 45–200 μm) (Harvey & Epifanio 1997; Davis et al. 2005a; Nghia
preference from rotifers to Artemia as food for Z3 larvae was also re-
et al. 2007a) and slow swimming movement make the rotifers,
ported in the Chinese mitten crab Eriocheir sinensis H. Milne Edwards,
Brachionus plicatilis (Müller, 1786) ideal first feed for mud crab larvae
1853 (Sui et al. 2008).
(Baylon et al. 2004; Baylon 2009). Artemia nauplii, with their slightly
larger size (2.7 μg and 428–517 μm) (Davis et al. 2005a) compared to
rotifers, are commonly used in the larviculture of mud crab as well 4.5.1.2. Copepods. Essentially the natural diet of various marine fish
(Baylon et al. 2004; Ruscoe et al. 2004; Davis et al. 2005b). Although and crustacean larvae (Okazaki et al. 2008; Takahashi & Uchiyama
unsuitable as food to early zoeal stages, they are proven to increase the 2008), copepods are starting to gain attention as alternative first live
growth and survival of larvae when fed at later larval stages (Baylon feed for the larviculture of many aquaculture species due to their
et al. 2004; Ruscoe et al. 2004). known benefits of improving the overall survival, growth and

45
K. Waiho et al. Aquaculture 493 (2018) 37–50

development of larvae (Imsland et al. 2006; Liu & Xu 2009; Barroso Table 8
et al. 2013; Karlsen et al. 2015). They are more superior in terms of Microbound diet composition used for S. serrata larvae and megalopae
nutritional values (higher protein, essential fatty acids, phospholipids (Genodepa et al. 2004a, 2004b, 2006, 2007; Holme et al. 2006).
and other micronutrients e.g. taurine, astaxanthin and zinc) (Karlsen Ingredient Dry weight (%)
et al. 2015) and digestibility compared to other commonly used live
feeds such as rotifers and Artemia (Pederson 1984; Hamre et al. 2013). Squid meal 39.7
Rotifer 39.7
Coupled with the broad spectrum of sizes in different life stages (i.e.
Fish oil 5.0
nauplii and copepodites) (Takahashi & Uchiyama 2008; Waiho et al. Corn oil 1.0
2013), copepods are excellent candidates to be used as live feed for first Lecithin 3.0
feeding of larvae. Cholesterol 1.0
Dibasic calcium phosphate (DCP) 0.6
Research on the use of copepods in crustacean larviculture, how-
Mineral mix 4.0
ever, are limited. It is still unconfirmed if copepods form the natural Vitamin mix 3.0
diet of mud crab larvae, although the carnivorous nature of Scylla Zein (binder) 3.0
larvae and the appropriate size of copepods makes them suitable pre- Total 100
dator-prey candidates. Using various combinations of rotifers, Artemia,
microencapsulated feed and frozen copepod, Jantrarotai et al. (2004)
found that S. olivacea larvae fed with a combination of either rotifer and sufficient nutrients to support the growth and development up to
Artemia or rotifer and frozen copepod gave the highest survival rates megalopae, the formulated microbound diet may be nutritionally su-
and fastest growth with development from Z1 to Z5 in 20.7 days and perior over Artemia, especially in enhancing the molting process. Larvae
21.7 days, respectively. Faster growth rate, greater length and weight fed with Artemia showed shorter development time and higher survival
gain, and higher astaxanthin content were observed when black tiger compared to those fed with microbound diet (Holme et al. 2006). In
shrimp, Penaeus monodon Fabricius, 1798 larvae were fed with three combination, however, a ratio of 1:1 microbound diet:Artemia resulted
types of copepods (Macrosetella gracilis (Dana, 1847), Pseudodiaptomus in the highest survival and development rate from Z3 to Z5 whereas
sp. and Oithona rigida Giesbrecht, 1896) compared to feeding with Ar- larvae fed with 100% microbound diet showed poor survival (Holme
temia nauplii alone (Santhanam et al. 2011). The enhanced growth et al. 2006). This highlights the potential of incorporating microbound
observed in copepod-fed crustacean larvae was also evident in marine diet into the dietary component of mud crab larviculture. Similar po-
fish species (Busch et al. 2010; Koedijk et al. 2010; Cassiano et al. 2011; sitive results of feeding larvae with a mixed diet of zooplankton and
Leu et al. 2012; Karlsen et al. 2015; de Melo-Costa et al. 2015). Thus, formulated diets have also been reported in other crustacean species,
future research on screening and selecting suitable copepods to be used including penaeid prawn (Jones et al. 1993) and lobster (Johnston et al.
as live feed for Scylla larvae could contribute significantly towards the 2008).
feed development of mud crab larviculture. When tested on S. serrata larvae of each stage (fed with microbound
One major hurdle of the potential usage of copepods as live feed in diet twice the equivalent dry weight of live feed), Genodepa et al.
mud crab larviculture, however, is the high costs involved in their mass (2004a) reported that microbound diet size preference of larvae in-
production and in obtaining sufficient copepods of a particular life creases as larvae develop. Further experimentation showed that the
stage at a specific time (Genodepa et al. 2004a; Conceição et al. 2010; ration for larvae was excessive as there was no significant difference
van der Meeren et al. 2014). Most studies had only produced copepods ration was reduced by 50%. By studying the radioactive contents hourly
at a pilot scale (Santhanam et al. 2011; Puello-Cruz et al. 2015). It is after S. serrata larvae was fed with 14C-labelled microbound diet,
therefore recommended to first develop a baseline performance curve Genodepa et al. (2006) suggest that the formulated microbound diet
using selected species of copepods as live feed for mud crab larvae was readily ingested by all larval stages, with an increase of > 90 folds
(Conceição et al. 2010). Once the appropriate species of copepods are in ingestion between Z1 and megalopae. The obvious increases in in-
identified, they may be incorporated into the daily larvae feed, or partly gestion were in Z3, Z5 and megalopa stages, indicating that the re-
together with rotifers or Artemia while their mass production protocols placement of live feed with microbound diets at the later larval stages
are being developed (Conceição et al. 1997). was feasible (Genodepa et al. 2006). The suitability of zein as binder
was also validated when Genodepa et al. (2007) compared with the
4.5.2. Microbound diet leaching rates of the same microbound diets bound with agar, alginate,
A main drawback of using live feed is their nutritional inconsistency carrageenan, gelatin or zein. The lowest leaching rate in zein-bound
which varies based on species, strain, age and culture techniques (Tizol- microbound diet is recommended as early and higher leaching rates of
Correa et al. 2006; Amarouayache et al. 2017; Broach et al. 2017). In microbound diets bounded with other binders decrease their nutritional
addition, live feeds are laborious and expensive to culture, and poten- values and could adversely influences the water quality (Genodepa
tially act as vectors for the introduction of pathogenic microbes et al. 2007).
(Genodepa et al. 2004a; Tizol-Correa et al. 2006; Hamre et al. 2013).
Thus, the development of formulated feeds that are applicable in the 5. Conclusions and recommendations
early larval stages could reduce the current dependency on live feed
and could lead to a more consistent hatchery production. Mud crab is regarded as a promising aquaculture species with its
Microbound diets are feeds produced by binding different compo- fast growth rate and high market demand (Fazhan et al. 2017a; Waiho
nents together with a binder, which are then dried, crushed and sieved et al. 2017). Significant progress has been made in the last decades to
to the required particle size (Holme et al. 2009b; Hamre et al. 2013). provide the optimum rearing parameters, to better understand the
Compared to live feed, microbound diets are storable, more convenient feeding mechanisms and nutritional requirements, and to develop sui-
in terms of required size and availability, and involve cheaper pro- table diets for Scylla larvae. Collection of newly hatched larvae should
duction costs (Southgate & Partridge 1998; Holme et al. 2009b). Using a be done within an hour after hatching through siphoning or scooping
formulated microbound diet formula (Table 8), Genodepa et al. (2004b) with the aid of light. Acclimatization is recommended if there is a water
showed that a 1:4 ratio of microbound diet to Artemia resulted in the temperature difference between the spawning tank and larval rearing
highest S. serrata megalopae survival. Furthermore, molting rate at both tanks. The optimum temperature and salinity range combination for
treatments (larvae were fed either 100% microbound diet, or purely larval rearing are 25–30 °C and 20–35 ppt respectively. An initial
Artemia) was high (90%) but those fed with microbound diet molted stocking density of 100–150 Z1/L, use of probiotics as prophylaxis and
earlier (Genodepa et al. 2004b). This suggests that in addition to having rearing in dark coloured tanks are proven to enhance the survival,

46
K. Waiho et al. Aquaculture 493 (2018) 37–50

growth and development of Scylla zoeae. Non-recirculating system acid composition of five Algerian bisexual and parthenogenetic strains of Artemia
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10.1007/s10499-017-0136-z.
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Ates, M.C.D., Quinitio, G.F., Quinitio, E.T., Sanares, R.C., 2012. Comparative study on the
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Busch, K.E.T., Falk-Petersen, I.-B., Peruzzi, S., Rist, N.A., Hamre, K., 2010. Natural zoo-
This work was supported by the Science and Technology Program of plankton as larval feed in intensive rearing systems for juvenile production of Atlantic
Shantou (No. 2016-44); Shantou University (STU) Scientific Research cod (Gadus morhua L.). Aquac. Res. 41, 1727–1740.
Cabello, F.C., 2006. Heavy use of prophylactic antibiotics in aquaculture: a growing
Foundation for Talents (No. NTF17006); the National Program for problem for human and animal health and for the environment. Environ. Microbiol. 8
Support of Top-Notch Young Professionals, China; the “Sail Plan” (7), 1137–1144.
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2016. Comparative efficacies of tilapia green water and biofloc technology (BFT) in
Province, China (14600702); and Niche Research Grant Scheme
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